Project Summary Spiral ganglion neurons (SGNs) encode everything an animal hears and send this information to the brain. In order to achieve rapid and reliable signal transmission, SGNs exhibit a number of specialized properties, including the ability to respond to glutamate via large, AMPA-receptor rich post-synaptic densities. Although all SGNs are glutamatergic, differences in the nature of their synapses and their responses to sound indicate that there are three distinct subtypes. High spontaneous firing rate (SR) SGNs have low thresholds and are likely the first to respond to sound. Low SR SGNs have higher thresholds and are proposed to improve the ability to detect sounds in noise; medium SR SGNs fall in between. These physiological differences are accompanied by parallel changes in the abundance of AMPA receptors and the size of the opposing pre-synaptic ribbon. Low SR SGN synapses are more vulnerable to the effects of noise exposure, which may be why some people have trouble understanding what they hear despite normal auditory thresholds. The long term goal of this project is to understand how SGNs acquire the properties needed for the perception of sound. More immediately, we will define the intrinsic transcriptional networks that endow SGN subtypes with their distinct properties and functions. We hypothesize that SGN diversification depends on the combined activities of a pan-SGN Gata3 network and a subtype-specific program driven by the transcription factor Runx1. Previously, we showed that Gata3 influences multiple features of SGN differentiation, acting in part through the transcription factor Mafb (Lu et al., 2011; Appler et al., 2013; Yu et al., 2013). In Mafb mutant mice, SGNs do not develop normal post-synaptic densities. Subsequently, we showed that Type I SGNs fall into three molecular distinct subtypes (Ia, Ib, and Ic) that match the features of high, medium, and low SR SGNs respectively (Shrestha et al., 2018). New preliminary studies suggest that diversification among Type I SGNs requires the transcription factor Runx1, which is restricted to Ib and Ic subtypes by late embryogenesis and then maintained throughout life. Further, Ic SGNs appear to be lost from Runx1 conditional knock-out (CKO) mice, as indicated by changes in gene expression and altered ABR responses. Here, we will define the role of Runx1 and its relationship with Gata3. We will perform a thorough analysis of Runx1CKO mice, examining SGN composition, synaptic heterogeneity, and the effects on hearing. In parallel, we will use genetic and viral overexpression approaches to learn how Gata3 and its Maf effectors influence the emergence of subtype identity and synaptic heterogeneity in vivo. Using single cell and bulk RNA-sequencing, we will define the molecular programs active in developing Ib/c SGNs and test how these programs are altered by loss of Runx1 or Gata3, as well as how the Maf factors contribute. These studies will elucidate the molecular programs driving SGN diversification, show how this diversity influences hearing, and may reveal a way to replace lost Ic SGNs and hence restore normal hearing after acoustic trauma.